Genome-wide identification, characterization and gene expression of BES1 transcription factor family in grapevine (Vitis vinifera L.)

BES1, as the most important transcription factor responsible for brassinolide (BR) signaling, has been confirmed to play a significant role in regulating plant growth and the improvement of stress resistance. The transcriptional regulatory mechanism of BES1 has been well elucidated in several plants, such as Arabidopsis thaliana (A. thaliana), Triticum aestivum L. (T. aestivum), and Oryza sativa L. (O. sativa). Nevertheless, the genome-wide analysis of the BES1 family in Vitis vinifera L. (V. vinifera). has not been comprehensively carried out. Thus, we have conducted a detailed analysis and identification of the BES1 transcription factors family in V. vinifera; a total of eight VvBES1 genes was predicted, and the phylogenetic relationships, gene structures, and Cis-acting element in their promoters were also analyzed. BES1 genes have been divided into three groups (I, II and III) based on phylogenetic relationship analysis, and most of VvBES1 genes were in group III. Also, we found that VvBES1 genes was located at seven of the total nineteen chromosomes, whereas VvBES1-2 (Vitvi04g01234) and VvBES1-5 (Vitvi18g00924) had a collinearity relationship, and their three copies are well preserved. In addition, the intron–exon model of VvBES1 genes were mostly conserved, and there existed several Cis-acting elements related to stress resistance responsive and phytohormones responsive in BES1s genes promoter. Moreover, the BES1 expressions were different in different V. vinifera organs, and BES1 expressions were different in different V. vinifera varieties under saline-alkali stress and heat stress, the expression of VvBES1 also changed with the prolongation of saline-alkali stress treatment time. The above findings could not only lay a primary foundation for the further validation of VvBES1 function, but could also provide a reference for molecular breeding in V. vinifera.

BES1 and BZR1 are two key transcription factors that were initially identified as specifically regulating BRs to mediate gene expression 10 . The two genes belong to a family of six genes, including VvBES1, VvBZR1, VvBEH1, VvBEH2, VvBEH3 and VvBEH4. It has been reported that BES1/BZR1 mediate BR signal transduction, and BES1/BZR1 could regulate plant growth and development by regulating the expression of downstream target genes in BR biosynthesis 11 . BES1 has been confirmed to have a significant impact on BR signal transduction, plays an important role in both abscisic acid (ABA) and strigolactone (SL) signaling 12 , and it could interact with REPRESSOR OF GA1-3 (RGA), a member of the DELLA transcriptional regulatory family, by regulating the biosynthesis of gibberellin 3 (GA 3 ), and co-controlling cell elongation and plant growth 13 . Moreover, the interaction of BES1 and BZR1 co-regulated plant physiological metabolism through different signaling pathways, thus regulated plant growth development, stress response formation, and promote the stable transmission of BR signal downstream through the dual regulation of phosphorylation, etc 14 . In recent years, BES1 interacts with BIM1 has been widely reported, which recognized as a basic helix-loop-helix protein, will bind to E box (CANNTG) sequences present in many promoters (BR-induced) 15 . And UV-B activated the expression of UVR8 and inhibits the DNA binding activity of BES1/BIM1, thus affecting the occurrence of photomorphogenesis in A. thaliana 16 . Further investigation illustrated that, BES1 and BZR1 mutant, showed BR-activated phenotypes (enhanced hypocotyl elongation). In contrast, BES1 and BIM mutant, indicated hypocotyl shortened and dwarf phenotypes 17 . It has been reported that there is an interaction between BES1 and other transcription factors, such as the PIF4, WRKY and MYB gene families 18 . For instance, the interaction between PIF4 and BES1 could control cell elongation and plant growth 19 , WRKY46, WRKY54, and WRKY70 interacts with BES1 co-regulate plant stress resistance formation 20 , and the interaction between MYB and BES1 regulates the accumulation of anthocyanins and the improvement of fruit quality 21 .
Vitis vinifera L., as a widely planted economic crop around the world, is recognized by consumers because of its sweet, sour taste, as well as its high nutritional value. In recent years, the genome-wide identification of BES1 has been well studied in A. thaliana and T. aestivum 22,23 , and some genome-wide identifications have also been carried out in horticultural crops including  [24][25][26][27][28][29] . However, the genome-wide identification and characterization, and phylogenetic relationship of the BES1 family has not been systematically studied in V. vinifera. There was one triplication event in V. vinifera ancestors. Theoretically, there should be a lot of duplicate genes with three copies in V. vinifera. However, during the evolution, some copies were lost while some were still retained 30 . It is also of great significance to explore the gain and loss of copies in VvBES1 genes. Thus, in this study, the functions of BES1 of V. vinifera were investigated, the phylogenetic relationship of BES1 gene family members were systematically analyze, the structure and promoter elements of BES1 genes were explored. Meanwhile, the interspecific collinearity analysis among Amborella trichopoda (A. trichopoda), V. vinifera and A. thaliana, the intraspecific collinearity analysis of VvBES1s of V. vinifera, and the search of copies of VvBES1 genes were furtherly conducted. To further explore the differential expression of VvBES1 genes in V. vinifera organs as well as their abilities to cope with various stresses, the expression pattern of the VvBES1 genes in different organs, various abiotic and biological stresses of V. vinifera were quantified. In all, the above findings could lay a primary foundation for understanding the potential regulatory mechanisms of BES1 in V. vinifera, and provide a reference for future V. vinifera breeding.
Firstly, at Shanghai Jiao Tong University, the roots, stems, leaves, inflorescences and berries of six "Muscat Hamburg" under conventional cultivation were collected for analysis. At 20 days after anthesis (DAA 20), six "Muscat Hamburg" were subjected to heat stress treatment (40 °C, 8 h) and another six "Muscat Hamburg" continued using normal cultivation. At the stages of green berry, verasion and maturity in both the treatment group and control group, thirty berries were collected randomly from the upper, middle and lower parts of the fruit cluster. At the same time, six "Muscat Hamburg" were subjected to saline-alkali stress treatment, and a 0.5% NaCl and 1% NaHCO 3 mixed solution (1 L) was applied to the V. vinifera every three days; we then chose another six V. vinifera, and used normal rain shelter cultivation for the control group. Thirty leaves were collected from each plant every five days from the beginning of treatment for further analysis.
Secondly, at Zhejiang Zhoushan Academy of Agriculture and Forestry Sciences Ma 'ao base (121°30′ E, 29°32′ W), at 20 days after anthesis (DAA 20), eighteen V. vinifera of each variety ("A17", "Benifuji", "Shine Muscat" and "Ruby Seedless") were selected; nine were used for saline-alkali treatment and nine were used for control. In saline-alkali treatment group, NaCl and NaHCO 3 (80 mmol/l) mixed solution (pH 8.5) was used to irrigate the vines every three days. In the control group, Hoagland's nutrient solution was used to irrigate the vines every 3 days. At maturity stage, 30 berries were collected from the upper, middle and lower parts of the fruit cluster from each treatment group, and immediately mailed back to Shanghai Jiao Tong University for further study.  Table S1.

Data analysis and figure drawing.
In the matter of data analysis, SPSS 16.0 (IBM, Armonk, NY, USA) was used for analyzing the difference significance of data, which was expressed in the form of mean + standard error (SE). Independent sample T-test, and univariate variance homogeneity of ANOVA were applied to analyze the difference significance among treatments (P < 0.05). GraphPad Prism 9.0 (GraphPad Software Inc., San Diego, CA, USA) was applied for histogram and heat map rendering.

Analysis of gene structure and conserved motifs in V. vinifera. Since the distribution of exons
and introns is also an aspect of the information of gene, we also analyzed the structural characteristics of eight VvBES1 genes, including the numbers and lengths of their exons and introns (Fig. 2). The results show that every VvBES1s had at least two exons, except for VvBES1-3. Moreover, we found that VvBES1-6 and VvBES1-7 had more introns than the other four VvBES1s genes. The evolution of VvBES1 was accompanied by posttranscriptional gene regulation and modification, resulting in the variation of its gene structures, which also indirectly indicates that BES1 structural differences would lead to the diversification of gene functions. Compared with VvBES1 genes, we found that all ATBES1 genes were conserved, with the lengths ranging from 1600 to 45,000 KB, and AT2G45880 contained more introns, while AT3G50750 contained fewer. Different gene structures would inevitably lead to differences in gene functions.
In order to better understand the diversity and similarities of VvBES1 proteins, as Fig. 3 has shown, there were ten kinds of motifs found in both V. vinifera and A. thaliana. Specifically, there were night kinds of motifs in VvBES1-1, seven kinds of motifs in VvBES1-2, nine kinds of motifs in VvBES1-3, two kinds of motifs in VvBES1-4, seven kinds of motifs in VvBES1-5, two kinds of motifs in VvBES1-6, two kinds of motifs in VvBES1-7, and two kinds of motifs in VvBES1-8 (Fig. 3A). Concerned with motifs in A. thaliana, there were seven kinds of motifs in AT1G19350, seven kinds of motifs in AT1G75080, six kinds of motifs in AT1G78700, six kinds of motifs in AT2G45880, seven kinds of motifs in AT3G50750, six kinds of motifs in AT4G18890, four kinds of motifs in AT4G36780, and six kinds of motifs in AT5G45300 (Fig. 3B). www.nature.com/scientificreports/ Cis-acting element analysis of VvBES1 promoters. In order to better understand the possible regulatory mechanism of the VvBES1 gene in V. vinifera growth and development, we studied the Cis-acting elements in the promoter of VvBES1 genes. As shown in Fig. 4, the Cis-acting elements, could be divided into stress-response element, light response element, phytohormones elements, and so on. To be specific, there were seven kinds of elements in VvBES1-1 (four for phytohormones, one for light response, one for stress response, and one for flavonoid biosynthesis), four kinds of elements in VvBES1-2 (two for phytohormones, one for light response and one for stress response), seven kinds of elements in VvBES1-3 (three for phytohormones, one for light response, two for stress response and one for MYB binding site), six kinds of elements in VvBES1-4 (four for phytohormones, one for light response and one for stress response), four kinds of elements in VvBES1-5 (one for phytohormones, one for light response, one for stress response and one for MYB binding), and six kinds of elements in VvBES1-6 (three for phytohormones, one for light responsiveness, and two for stress response), six kinds of elements in VvBES1-7 (four for phytohormones, and one for stress response, and one for MYB binding), and eight kinds of elements in VvBES1-8 (four for phytohormones, and one for stress response, one     Structure prediction of VvBES1 proteins. The structure of a protein determines its biological function. Further analysis of the tertiary structure of the protein indicated that VvBES1 was further coiled and folded on the basis of the secondary structure, forming a tertiary structure maintained by the secondary bonds, on which there were many hydrophobic bonds, salt bonds and hydrogen bonds (Fig. S1). Moreover, the protein structure of VvBES1-7 was different from those of the other seven BES1 genes, which confirms that it might have different biological functions from other BES1s genes.
Synteny analysis of BES1 gene family. In order to further understand the evolution of BES1 gene family, the interspecific and intraspecific collinearity analysis of this gene family were also carried out in this study. For the interspecific analysis of BES1, we selected A. thaliana (model plants), A. trichopoda (the earliest known angiosperms that evolved separately from other angiosperms) and V. vinifera as research object, to test the consistency of their gene sequence. Figure 5A indicates that a total of four pairs of BES1 genes had collinear relationships in V. vinifera and A. trichopoda (VvBES1-6 and AMTR_s00011p00265640; VvBES1-5 and AMTR_ s00059p00136790; VvBES1-1 and AMTR_s00170p00061300; VvBES1-3 and AMTR_s00170p00061300). And seven pairs of BES1 genes had collinear relationships in V. vinifera and A. thaliana (VvBES1-1 and AT4G18890; VvBES1-5 and AT1G19350; VvBES1-5 and AT1G75080; VvBES1-5 and AT3G50750; VvBES1-6 and AT5G45300; VvBES1-2 and AT3G50750; VvBES1-5 and AT4G36780). Furthermore, for the interspecific analysis of BES1 in V. vinifera, as shown in Fig. 5B, we found there were many colinear gene pairs in the whole V. vinifera genome, but we only found that VvBES1 genes, located on chromosome 18 (VvBES1-5) had a collinearity relationship with the genes located on chromosome 4 (VvBES1-2). Finally, we searched known genes in the obtained synteny list for WGD analysis in Vitis Intraspecific. The results demonstrated that in the process of V. vinifera evolution, the genes with three copies of VvBES1-2, VvBES1-5, and VvBES1-6 were completely retained, while genes with only two copies were retained in VvBES1-1 and VvBES1-3, while there was no evolutionary relationship in VvBES1-4, VvBES1-7 and VvBES1-8 (Fig. 5C).
Expression patterns of VvBES1 in diverse organs of V. vinifera. In order to explore the possible biological functions of VvBES1, the expression levels of eight VvBES1s in V. vinifera roots, stems, leaves, inflorescences and berries were determined by qRT-PCR. As Fig. 6 has shown, VvBES1-1 and VvBES1-2 were expressed at high levels in both the stem and the root (Fig. 6A,B), and the expression of VvBES1-3 was higher in the stem, inflorescence, leaf and root (Fig. 6C), while the expression of VvBES1-4 was higher in both stem and inflorescence (Fig. 6D). Moreover, VvBES1-5, VvBES1-6, VvBES1-7 and VvBES1-8 were expressed at high levels in the inflorescence (Fig. 6E-H). It is worth noting that the expression levels of all the VvBES1 genes in V. vinifera berries were very low, while they were generally high in leaves and inflorescences.   Fig. 7 indicate that the expressions of VvBES1-1, VvBES1-2, VvBES1-3 and VvBES1-4 were lower and then higher. At the green berry stage, heat stress treatment increased VvBES1-1, VvBES1-2, and VvBES1-4 expressions (Fig. 7A,B,D), while the expression level of VvBES1-3 was upregulated in the control group (Fig. 7C). The expression levels of VvBES1-5 and VvBES1-8 in the green berry and in the veraison stages, heat stress treatment increased their expressions (Fig. 7E,H). Interestingly, the variation tendency of VvBES1-6 expression increased and then decreased in the heat stress treatment group, decreased and then increased in the control group, heat stress up-regulated expression of VvBES1-6 at the veraison stage (Fig. 7F). Moreover, the expression of VvBES1-7 decreased then increased, while in the control group, and the expression of VvBES1-7 was significantly up-regulated at the maturity stage (Fig. 7G).

Expression patterns of VvBES1 under saline-alkali stress. The expression patterns of VvBES1 under
saline-alkali stress in different V. vinifera varieties were also investigated in this study. As the results shown in the Fig. 8, by detecting the expression levels of VvBES1 in ripening berries in different V. vinifera varieties under saline-alkali stress, we found that all BES1 genes were expressed at a high level in "A17" while expressed a low level in "Benifuji", except for VvBES1-3, VvBES1-4 and VvBES1-7 (Fig. 8A-H).   (VvBES1-1 of berry), which are represented as log 2 fold change. The one-way Duncan's new multiple-range test was implemented to assess the significance of differences among treatments (p < 0.05).
(I) The expression profiles of VvBES1 gene under saline-alkali stress in "Muscat Hamburger" V. vinifera leaves; the error bars represent ± SE (n = 3), and the relative expression changes are relative to the VvBES1-1 at 5 days after treatment, which are represented as log 2 fold change. The one-way Duncan's new multiple-range test was implemented to assess the significance of differences among treatments (p < 0.05).

Discussion
The BES1 gene family has previously been reported in a variety of horticulture plants, including M. pumila, A. chinensis, C. sativus, S. lycopersicum, S. tuberosum, C. reticulata and M. nana [24][25][26][27][28][29]38,39 . Before now, the transcriptional regulation mechanism of the BES1 transcription factor had been effectively revealed in A. thaliana, O. sativa and T. aestivum 22,25 A. trichopoda, the evolutionary tree shows that the BES1 gene family could be divided into three groups in each, and most of the VvBES1 genes were in group III. Previous studies have reported that a change of in gene family size might be beneficial, harmful or neutral, but changes in gene family number are also one of the most important reasons for species specificity 40 . Our results further support the conclusion, determined that the genetic relationship of BES1 gene in different species was different, and the BES1 genes in V. vinifera were more closely related to Prunus, A. chinensis and A. thaliana. This had important implications for future studies of their functions and mechanisms of transcriptional regulation. Further studying protein tertiary structure and subcellular localization, we found there were a lot of α-helices and random coils in VvBES1 protein, whereas they essentially located in cytoplasmic and nuclei. Therefore, we could make a reasoned that VvBES1 would fulfil a role in their biological functions in the cytoplasm and nucleus, and different function was decided by different protein structure.
The Cis-acting elements included promoters, enhancers, regulatory sequences and inducible elements, which could affect gene expression and regulate gene expression, ultimately leading to differences in plant growth and development 41 . In this study, several kinds of Cis-acting elements related to stress and phytohormones were found located in the VvBES1s promoters. Nevertheless, the numbers of these elements varied in different VvBES1 proteins, ultimately resulting in the different biological functions of VvBES1 in relation to regulating the growth and development of V. vinifera. As regards GA responsive, it was previously reported that GA signal transduction interacted with the BR signal, while the DELLA protein could reduce AtBZR1 abundance and transcription level via participation in GA signal transduction 42 . The phylogenetic analysis also showed that AT5G45300 and VvBES1-6 were all in Group III, and AT2G45880 and VvBES1-7 were all in Group I, which indicates that VvBES1-6 and VvBES1-7 might be closer to the BES1 transcription factors in A. thaliana, and might have similar biological functions, which provides relevant reference for the further study that the functions of these two transcription factors. Furthermore, auxin response factor (ARF) was the key transcription factor regulating the expressions of several auxin response genes 43 . It has been confirmed that a variety of ARF interact with BZR1 to regulate a series of activities, such as the growth and development of plants through signal cross talk with BR 44 . Our results showed that there existed IAA responsive elements in VvBES1-1, VvBES1-3, VvBES1-4, VvBES1-6, VvBES1-7 and VvBES1-8, which further corroborated that BR signal might have crosstalk with IAA signal in V. vinifera. In regard with stress resistance elements in VvBES1, we found many low-temperature responsive elements in VvBES1-2 and VvBES1-3, and many stress-related responsive elements in VvBES1-1, VvBES1-4, VvBES1-5, VvBES1-6, VvBES1-7 and VvBES1-8. In addition, VvBES1-2, which possessed more low-temperature responsive elements were in the same group as AT3G50750, AT1G19350, AT4G18890, AT1G75080, AT5G45300, AT4G36780, and AT1G78700, further confirmed the previous finding demonstrated BES1 is closely related to the formation of frost resistance in A. thaliana 45 . VvBES1-7, which owned more stress-related responsive elements were in the same group as AT2G45880, which was also consistent with a previous study's finding that ATBES1 was closely associated with the improvement of stress resistance 10 . The diagram of the tertiary structure pattern of the protein also showed much random crimping in protein structure, which was beneficial to the formation of their biological functions. Collinearity studies the arrangement of homologous genes or sequences. It is more common to evaluate the assembly effect of genomes and the retention and loss of homologous genes, through homology comparisons, to study the evolutionary relationships of diverse materials. Through further colinear analyses and evolutionary relationship exploration of BES1 gene family, we found four pairs of BES1 genes had collinear relationships in V. vinifera and A. trichopoda, and there existed seven pairs of collinear BES1 genes in V. vinifera and A. thaliana. Also, in V. vinifera genome, many colinear gene pairs were found, but only two VvBES1 genes were highly collinear, and located on different chromosomes (4, 18, respectively). Moreover, we found copies of only three VvBES1 genes (VvBES1-2, VvBES1-5, and VvBES1-6) have been well preserved, two (VvBES1-1and VvBES1-3) have lost one copy, while three (VvBES1-4, VvBES1-7 and VvBES1-8) have no evolutionary relationship. Therefore, we have inferred that gene pairs with collinearity might have the analogous biological functions, while genes without collinearity could regulate the production of different traits and physiological activities in plants due to their different biological functions. Since there is a collinear relationship between VvBES1-2 and VvBES1-5, and their copies have been completely preserved, it is meaningful to extensively study their biological functions in the future.
BR, a plant hormone that was found in recent decades, and has been called the sixth hormone after IAA, cytokinin, GA, ABA and ethylene 46 . BR is tightly involved in various stress responses and the increase in stress resistance 47 . Therefore, the BR signal's regulation of the key transcription factor BES1 has been demonstrated to be involved in plant stress response, such as water stress, heat damage, cold damage, etc 23,48,49 . To further study the role of the VvBES1 gene in plant responses to stress, we have also carried out several quantitative experiments to characterize the expression profiles of the VvBES1 gene in different V. vinifera organs, and the their capacity in response to heat stress and saline-alkali stress. In all, the expressions of different kinds of VvBES1 proteins are all higher in stems, roots and inflorescence, and VvBES1s could respond differently to heat stress www.nature.com/scientificreports/ and saline-alkali stress. Specifically, VvBES1-1 and VvBES1-2 genes showed relatively consistent change trends, whereas heat stress significantly up-regulated their expressions. Under the heat stress, the increases in the expression level of VvBES1s mainly occurred in the veraison stage and the green berry stage, indirectly proving that VvBES1 is an important transcription factor in response to heat stress at early fruit ripening stage. In terms of saline-alkali stress, we found "A17" was more drought-tolerant than other varieties, and with the extension of treatment time, the VvBES1-1, VvBES1-4 and VvBES1-8 genes showed a gradual upward trend. The above findings illustrated that all VvBES1 proteins might participate in responses to stress, though the expression levels of BES1 were also different in diverse V. vinifera varieties and different organs, the up-regulation of most of them could positively regulate the improvement of V. vinifera stress resistance. This assertion is verified by studies using A. thaliana and O. sativa as material, confirming that BES1 played an important role in regulating the development of stress resistance and phytohormones-related activities 50 . In addition, the interaction between BES1 and several transcription factors are also been widely reported. At present, it is known that BES1/BZR1 family transcription factors can affect plant growth and development through a brassinosteroid-dependent pathway and brassinosteroid-independent pathway 51 . Relevant studies have shown that CRY1 not only interacted with BIM1 physically, but also interacted with BES1 in a blue light dependent manner, as which the three together regulated the elongation of A. thaliana hypocotyls 50 . It is a pity that our study did not continue to explore whether VvBES1 gene interacts with VvBZR1 or VvBIM1 in regulating the improvement of V. vinifera stress resistance or the occurrence of phytohormones responses. However, our experimental results still systematically summarized the basic structure and possible biological functions of VvBES1. As well, we have attempted to clarify the evolutional and collinearity relationships of the VvBES1 gene family, thus laid a theoretical foundation for the development of subsequent molecular experiments.

Conclusion
In this study, we not only analyzed the basic structures of VvBES1 genes, but also studied their phylogenetic relationships, identified the motifs they embodied, and analyzed their chromosomal synteny and evolutionary relationship among Interspecific and intraspecific. Finally, we obtained the expression profiles of VvBES1 in different V. vinifera organs and diverse varieties, and the ability of VvBES1 to cope with heat stress and salinealkali stress was also assessed. In summary, different VvBES1 genes had different biological functions due to their different structures; during the continuous evolution of V. vinifera, copies of some VvBES1 gene were well retained, while some losses occurred in other VvBES1 gene; More BES1 collinear gene pairs were discovered in the V. vinifera vs. A. thaliana than in the V. vinifera vs. A. trichopoda. In addition, VvBES1 played significant role in the improvement of stress resistance, and their expressions in V. vinifera were not only tissue-specific, but also slightly differed under heat stress and saline-alkali stress. This study provides a reference for further studies on the function and transcriptional regulation mechanism of VvBES1, clarifies the degree of VvBES1s gene respond to stress, so as to speculate the specific role of VvBES1 in plant growth, development and stress resistance formation.